首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 125 毫秒
1.
The electron-transfer reaction between spinach wild-type plastocyanin (Pc(WT)) two site-directed mutants, Pc(Thr79His) and Pc(Lys81His), and spinach Photosystem 1 particles, has been studied as a function of protein concentration, ionic strength and pH by using laser-flash absorption spectroscopy. The kinetic data are interpreted using the simplest possible three-step model, involving a rate-limiting conformational change preceding intracomplex electron transfer. The three proteins show similar concentration, pH and ionic strength dependencies. The effects of ionic strength and pH on the reaction indicate a strong influence of complementary charges on complex formation and stabilization. Studies with apoprotein support the opinion that the hydrophobic patch is critical for an productive interaction with the reaction center of Photosystem 1. Together with earlier site-directed mutagenesis studies, the absence of a detectable Photosystem 1 reaction in the presence of reduced azurin, stellacyanin, cytochrome c and cytochrome c551, demonstrates the existence of a high level of specificity in the protein-protein interface in the formation of an efficient electron-transfer complex.  相似文献   

2.
A spinach plastocyanin (Pc) mutant, Pc(Leu12His), has been constructed by site-directed mutagenesis and expressed in Escherichia coli to probe the importance of the hydrophobic patch in the interaction with Photosystem 1. The mutant has been characterized by optical absorption, EPR spectroscopy and redox titration. The electron transfer to Photosystem 1 was investigated by flash-induced time-resolved absorption measurements at 830 nm. The Pc(Leu12His) mutant showed a major change in the Photosystem 1 kinetics compared to wild-type Pc. In contrast to the biphasic Photosystem 1 reduction observed for the physiological reaction partner, only the slow phase was discerned when Pc(Leu12His) replaced wild-type Pc as the electron donor. The reaction showed a hyperbolic dependence with increasing Pc concentration, saturating at a rate constant value of 2000 s-1, which is about 10 times slower than the corresponding rate constant for wild-type Pc. Obviously, this position i s critical for a proper reaction. Moreover, the reaction showed a titrating behavior with a pKa of 6.7. Thus, it appears that both shape and charge of the residue in this position are important. A plausible reaction mechanism for electron transfer between wild-type Pc and Photosystem 1 is discussed. The role of electrostatic interactions may be that of long-range guidance and initial recognition that allow the two proteins to seek a pre-docking configuration(s). Then a short-range rearrangement(s), involving also hydrophobic interactions, forms an optimum docking configuration prior to electron transfer.  相似文献   

3.
Plastocyanin (Pc) is a copper-containing protein, which functions as an electron carrier between the cytochrome b(6)f and photosystem 1 (PS1) complexes in the photosynthetic electron transfer (ET) chain. The ET is mediated by His87 situated in the hydrophobic surface in the north region of Pc. Also situated in this region is Leu12, which mutated to other amino acids severely disturbs the ET from cytochrome f and to PS1, indicating the importance of the hydrophobic surface. The crystal structure of the Pc double mutant G8D/L12E has been determined to 2.0 A resolution, with a crystallographic R-factor of 18.3% (R(free)=23.2%). A comparison with the wild-type structure reveals that structural differences are limited to the sites of the mutations. In particular, there is a small but significant change in the hydrophobic surface close to His87. Evidently, this leads to a mismatch in the reactive complex with the redox partners. For PS1 this results in a 20 times weaker binding and an eightfold slower ET as determined by kinetic measurements. The mutations that have been introduced do not affect the optical absorption spectrum. However, there is a small change in the EPR spectrum, which can be related to changes in the copper coordination geometry.  相似文献   

4.
Recombinant human myoglobin mutants with the distal His residue (E7, His64) replaced by Leu, Val, or Gln residues were prepared by site-directed mutagenesis and expression in Escherichia coli. Electronic and coordination structures of the ferric heme iron in the recombinant myoglobin proteins were examined by optical absorption, EPR, 1H NMR, magnetic circular dichroism, and x-ray spectroscopy. Mutations, His-->Val and His-->Leu, remove the heme-bound water molecule resulting in a five-coordinate heme iron at neutral pH, while the heme-bound water molecule appears to be retained in the engineered myoglobin with His-->Gln substitution as in the wild-type protein. The distal Val and distal Leu ferric myoglobin mutants at neutral pH exhibited EPR spectra with g perpendicular values smaller than 6, which could be interpreted as an admixture of intermediate (S = 3/2) and high (S = 5/2) spin states. At alkaline pH, the distal Gln mutant is in the same so-called "hydroxy low spin" form as the wild-type protein, while the distal Leu and distal Val mutants are in high spin states. The ligand binding properties of these recombinant myoglobin proteins were studied by measurements of azide equilibrium and cyanide binding. The distal Leu and distal Val mutants exhibited diminished azide affinity and extremely slow cyanide binding, while the distal Gln mutant showed azide affinity and cyanide association rate constants similar to those of the wild-type protein.  相似文献   

5.
Plastocyanin (Pc) has been modified by site-directed mutagenesis at two separate electron-transfer (ET) sites: Leu-12-Glu at a hydrophobic patch, and Tyr-83-His at an acidic patch. The reduction potential at pH 7.5 is decreased by 26 mV in Pc(Leu-12-Glu) and increased by 35 mV in Pc(Tyr-83-His). The latter mutant shows a 2-fold slower intracomplex ET to photosystem I (PSI) as expected from the decreased driving force. The affinity for PSI is unaffected for this mutant but is drastically decreased for Pc(Leu-12-Glu). It is concluded that the hydrophobic patch is more important for the ET to PSI.  相似文献   

6.
Gong XM  Paddock ML  Okamura MY 《Biochemistry》2003,42(49):14492-14500
The structure of the complex between cytochrome c(2) (cyt) and the photosynthetic reaction center (RC) from Rhodobacter sphaeroides shows contacts between hydrophobic residues Tyr L162, Leu M191, and Val M192 on the RC and the surface of the cyt [Axelrod et al. (2002) J. Mol. Biol. 319, 501-515]. The role of these hydrophobic residues in binding and electron transfer was investigated by replacing them with Ala and other residues. Mutations of the hydrophobic residues generally resulted in relatively small changes in the second-order electron-transfer rate k(2) (Br?nsted coefficient, alpha( )()= 0.15 +/- 0.05) indicating that the transition state for association occurs before short-range hydrophobic contacts are established. Larger changes in k(2), found in some cases, were attributed to a change in the second-order mechanism from a diffusion controlled regime to a rapidly reversible binding regime. The association constant, K(A), of the cyt and the rate of electron transfer from the bound cyt, k(e), were both decreased by mutation. Replacement of Tyr L162, Leu M191, or Val M192 by Ala decreased K(A) and k(e) by factors of 130, 10, 0.6, and 120, 9, 0.6, respectively. The largest changes were obtained by mutation of Tyr L162, showing that this residue plays a key role in both binding and electron transfer. The binding affinity, K(A), and electron-transfer rate, k(e) were strongly correlated, showing that changes of hydrophobic residues affect both binding and electron transfer. This correlation suggests that changes in distance across hydrophobic interprotein contacts have similar effects on both electron tunneling and binding interactions.  相似文献   

7.
Conserved phenylalanine 35 is one of the hydrophobic patch residues on the surface of cytochrome b5 (cyt b5). This patch is partially exposed on the surface of cyt b5 while its buried face is in direct van der Waals' contact with heme b. Residues Phe35 and Phe/Tyr74 also form an aromatic channel with His39, which is one of the axial ligands of heme b. By site-directed mutagenesis we have produced three mutants of cyt b5: Phe35-->Tyr, Phe35-->Leu, and Phe35-->His. We found that of these three mutants, the Phe35-->Tyr mutant displays abnormal properties. The redox potential of the Phe35-->Tyr mutant is 66 mV more negative than that of the wild-type cyt b5 and the oxidized Phe35-->Tyr mutant is more stable towards thermal and chemical denaturation than wild-type cyt b5. In this study we studied the most interesting mutant, Phe35-->Tyr, by X-ray crystallography, thermal denaturation, CD and kinetic studies of heme dissociation to explore the origin of its unusual behaviors. Analysis of crystal structure of the Phe35-->Tyr mutant shows that the overall structure of the mutant is basically the same as that of the wild-type protein. However, the introduction of a hydroxyl group in the heme pocket, and the increased van der Waals' and electrostatic interactions between the side chain of Tyr35 and the heme probably result in enhancement of stability of the Phe35-->Tyr mutant. The kinetic difference of the heme trapped by the heme pocket also supports this conclusion. The detailed conformational changes of the proteins in response to heat have been studied by CD for the first time, revealing the existence of the folding intermediate.  相似文献   

8.
Kageyama T 《Biochemistry》2004,43(48):15122-15130
Proteolytic specificities of human pepsin A and monkey chymosin were investigated with a variety of oligopeptides as substrates. Human pepsin A had a strict preference for hydrophobic/aromatic residues at P'1, while monkey chymosin showed a diversified preferences accommodating charged residues as well as hydrophobic/aromatic ones. A comparison of residues forming the S'1 subsite between mammalian pepsins A and chymosins demonstrated the presence of conservative residues including Tyr(189), Ile(213), and Ile(300) and group-specific residues in the 289-299 loop region near the C terminus. The group-specific residues consisted of hydrophobic residues in pepsin A (Met(289), Leu/Ile/Val(291), and Leu(298)) and charged or polar residues in chymosins (Asp/Glu(289) and Gln/His/Lys(298)). Because the residues in the loop appeared to be involved in the unique specificities of respective types of enzymes, site-directed mutagenesis was undertaken to replace pepsin-A-specific residues by chymosin-specific ones and vice versa. A yeast expression vector for glutathione-S-transferase fusion protein was newly developed for expression of mutant proteins. The specificities of pepsin-A mutants could be successfully altered to the chymosin-like preference and those of chymosin mutants, to pepsin-like specificities, confirming residues in the S'1 loop to be essential for unique proteolytic properties of the enzymes. An increase in preference for charged residues at P'1 in pepsin-A mutants might have been due to an increase in the hydrogen-bonding interactions. In chymosin mutants, the reverse is possible. The changes in the catalytic efficiency for peptides having charged residues at P'1 were dominated by k(cat) rather than K(m) values.  相似文献   

9.
We previously proposed the hydrophobic and bulky residues of the three loops, designated stereochemistry gate loops (SGLs), to constitute a hydrophobic substrate binding pocket of -hydantoinase from Bacillus stearothermophilus SD1. Simulation of substrate binding in the active site of -hydantoinase and sequence alignment of various -hydantoinases revealed the critical hydrophobic residues closely located around the exocyclic substituent of substrate. To evaluate the roles of these residues in substrate binding pocket, site-directed mutagenesis was performed specifically for Leu 65, Tyr 155, and Phe 159. When Tyr 155 was mutated to Phe and Glu, both mutants Y155F and Y155E were totally inactive for nonsubstituted hydantoin and -5-hydroxyphenyl hydantoin (HPH), which indicates that Tyr 155 is involved in substrate binding via a hydrogen bond with the hydantoinic ring. Furthermore, replacement of the hydrophobic residues Leu 65 and Phe 159 with Glu, a charged amino acid, resulted in a significant decrease in activity for nonsubstituted hydantoin, but not for HPH. The Kcat values of both mutants for nonsubstituted hydantoin also severely decreased, but a slight change in the Kcat values was observed towards HPH. These results suggest that the hydrophobic residues in SGLs play an essential role in substrate binding, and differentially interact according to the property of the exocyclic substituent.  相似文献   

10.
We have here investigated the dissociation kinetics of the His side chains axially ligated to the heme-iron in the ferric (1–56 residues) N-fragment of horse cyt c. The ligand deligation induced by acidic pH-jump occurs as a biexponential process with different pre-exponential factors, consistent with a structural heterogeneity in solution and the presence of two differently coordinated species. In analogy with GuHCl-denatured cyt c, our data indicate the presence in solution of two ferric forms of the N-fragment characterized by bis-His coordination, as summarized in the following scheme: His18–Fe(III)–His26 \rightleftharpoons His18–Fe(III)–His33. We have found that the pre-exponential factors depend on the extent of the pH-jump. This may be correlated with the different pKa values shown by His26 and His33; due to steric factors, His26 binds to the heme–Fe(III) less strongly than His33, as recently shown by studies on denatured cyt c. Interestingly, the two lifetimes are affected by temperature but not by the extent of the pH-jump. The lower pKa for the deligation reaction required the use of an improved laser pH-jump setup, capable of inducing changes in H+ concentration as large as 1 mM after the end of the laser pulse. For the ferric N-fragment, close activation entropy values have been determined for the two histidines coordinated to the iron; this result significantly differs from that for GuHCl-denatured cyt c, where largely different values of activation entropy were calculated. This underlines the role played by the missing segment (residues 57–104) peptide chain in discriminating deligation of the nonnative His from the sixth coordination position of the metal.  相似文献   

11.
The role played by the residues Leu12 and Lys33 – which are both located at the north hydrophobic patch of plastocyanin – in the interaction of the copper protein with Photosystem I from the cyanobacterium Synechocystis sp. PCC 6803 has been investigated by site-directed mutagenesis. A thermodynamic analysis of PS I reduction by wild-type and mutant plastocyanins has been performed by laser-flash absorption spectroscopy. In all cases, the electron transfer is impaired by mutations, which induce drastic changes in the apparent activation entropy of the overall reaction. Substitution of Leu12 by alanine specifically affects the hydrophobic interactions with PS I, whereas replacement of Lys33 by glutamate not only induces local electrostatic changes, but also alters the hydrophobic interactions with the photosystem. The thermodynamic analysis of the reactivity of K33E mutant towards PS I reveals that the effect of the mutation can be reversed by addition of magnesium cations, which probably bind at a place close to Glu33. The electrostatic surface potential does thus modulate the hydrophobic interactions with PS I by altering the solvent accessibility of some surface residues. This revised version was published online in June 2006 with corrections to the Cover Date.  相似文献   

12.
Chung-Hsien Hung 《BBA》2007,1767(6):686-693
Cytochrome (cyt) b559 has been proposed to play an important role in the cyclic electron flow processes that protect photosystem II (PSII) from light-induced damage during photoinhibitory conditions. However, the exact role(s) of cyt b559 in the cyclic electron transfer pathway(s) in PSII remains unclear. To study the exact role(s) of cyt b559, we have constructed a series of site-directed mutants, each carrying a single amino acid substitution of one of the heme axial-ligands, in the cyanobacterium Synechocystis sp. PCC6803. In these mutants, His-22 of the α or the β subunit of cyt b559 was replaced with either Met, Glu, Tyr, Lys, Arg, Cys or Gln. On the basis of oxygen-evolution and chlorophyll a fluorescence measurements, we found that, among all mutants that were constructed, only the H22Kα mutant grew photoautotrophically, and accumulated stable PSII reaction centers (∼ 81% compared to wild-type cells). In addition, we isolated one pseudorevertant of the H22Yβ mutant that regained the ability to grow photoautotrophically and to assemble stable PSII reaction centers (∼ 79% compared to wild-type cells). On the basis of 77 K fluorescence emission measurements, we found that energy transfer from the phycobilisomes to PSII reaction centers was uncoupled in those cyt b559 mutants that assembled little or no stable PSII. Furthermore, on the basis of immunoblot analyses, we found that in thylakoid membranes of cyt b559 mutants that assembled little or no PSII, the amounts of the D1, D2, cyt b559α and β polypeptides were very low or undetectable but their CP47 and PsaC polypeptides were accumulated to the wild-type level. We also found that the amounts of cyt b559β polypeptide were significantly increased (larger than two folds) in thylakoid membranes of cyt b559 H22YβPS+ mutant cells. We suspected that the increase in the amounts of cyt b559 H22YβPS+ mutant polypeptides in thylakoid membranes might facilitate the assembly of functional PSII in cyt b559 H22YβPS+ mutant cells. Moreover, we found that isolated His-tagged PSII particles from H22Kα mutant cells gave rise to redox-induced optical absorption difference spectra of cyt b559. Therefore, our results concluded that significant fractions of H22Kα mutant PSII particles retained the heme of cyt b559. Finally, this work is the first report of cyt b559 mutants having substitutions of an axial heme-ligands that retain the ability to grow photoautotrophically and to assemble stable PSII reaction centers. These two cyt b559 mutants (H22Kα and H22YβPS+) and their PSII reaction centers will be very suitable for further biophysical and biochemical studies of the functional role(s) of cyt b559 in PSII.  相似文献   

13.
The electron-transfer reactions of site-specific mutants of the blue copper protein azurin from Pseudomonas aeruginosa with its presumed physiological redox partners cytochrome c551 and nitrite reductase were investigated by temperature-jump and stopped-flow experiments. In the hydrophobic patch of azurin Met44 was replaced by Lys, and in the His35 patch His35 was replaced by Phe, Leu and Gln. Both patches were previously thought to be involved in electron transfer. 1H-NMR spectroscopy revealed only minor changes in the three-dimensional structure of the mutants compared to wild-type azurin. Observed changes in midpoint potentials could be attributed to electrostatic effects. The slow relaxation phase observed in temperature-jump experiments carried out on equilibrium mixtures of wild-type azurin and cytochrome c551 was definitively shown to be due to a conformational relaxation involving His35. Analysis of the kinetic data demonstrated the involvement of the hydrophobic but not the His35 patch of azurin in the electron transfer reactions with both cytochrome c551 and nitrite reductase.  相似文献   

14.
Adrenal cytochrome b561 (cyt b561), a transmembrane protein that shuttles reducing equivalents derived from ascorbate, has two heme centers with distinct spectroscopic signals and reactivity towards ascorbate. The His54/His122 and His88/His161 pairs furnish axial ligands for the hemes, but additional amino acid residues contributing to the heme centers have not been identified. A computational model of human cyt b561 (Bashtovyy, D., Berczi, A., Asard, H., and Pali, T. (2003) Protoplasma 221, 31-40) predicts that His92 is near the His88/His161 heme and that His110 abuts the His54/His122 heme. We tested these predictions by analyzing the effects of mutations at His92 or His110 on the spectroscopic and functional properties. Wild type cytochrome and mutants with substitutions in other histidine residues or in Asn78 were used for comparison. The largest lineshape changes in the optical absorbance spectrum of the high-potential (bH) peak were seen with mutation of His92; the largest changes in the low-potential (bL) peak lineshape were observed with mutation of His110. In the EPR spectra, mutation of His92 shifted the position of the g = 3.1 signal (bH) but not the g = 3.7 signal (bL). In reductive titrations with ascorbate, mutations in His92 produced the largest increase in the midpoint for the bH transition; mutations in His110 produced the largest decreases in ΔA561 for the bL transition. These results indicate that His92 can be considered part of the bH heme center, and His110 part of the bL heme center, in adrenal cyt b561.  相似文献   

15.
The role of the HELLGH (residues 450-455) motif in the sequence of rat dipeptidyl peptidase III (EC 3.4.14.4) was investigated by replacing Glu451 with an alanine or an aspartic acid residue and by replacing His450 and His455 with a tyrosine residue by site-directed mutagenesis. Mutated cDNAs were expressed three or four times in Escherichia coli, and the resulting proteins were purified to apparent homogeneity. None of the expressed mutated proteins exhibited DPP III activity. The mutants of Glu451 contained 1 mol of zinc per mole of protein, but mutants His450 and His455 did not contain significant amounts of zinc as determined by atomic absorption spectrometry. The Leu453-deleted enzyme (having the zinc aminopeptidase motif HExxH-18-E) had almost the same order of binding affinity (for Arg-Arg-2-naphthylamide) as the wild-type enzyme, but the specificity constant was about 10%. These results provide evidence that the suitable number of amino acids included between Glu451 and His455 is three residues for the enzyme activity and confirm that residues His450, His455, and Glu451 are involved in zinc coordination and catalytic activity.  相似文献   

16.
Two mutants of plastocyanin have been constructed by site-directed mutagenesis in spinach and pea to elucidate the binding and electron transfer properties between plastocyanin and spinach Photosystem 1. The conserved, surface-exposed Tyr-83 has been replaced by phenylalanine and leucine in plastocyanin from both species and the proteins have been expressed in Escherichia coli. The reaction mechanism of electron transfer from plastocyanins to photooxidized P700 in Photosystem 1 has been studied by laser-flash absorption spectroscopy. The experimental data were interpreted with a model involving a rate-limiting conformational change, preceding the intracomplex electron transfer. The pea proteins show an overall facilitated reaction with spinach Photosystem 1, compared to spinach plastocyanins. The changes are small but significant, indicating a more efficient electron transfer within the transient complex. In addition, for the spinach leucine mutant, the equilibrium within the plastocyanin-Photosystem 1 complex is more displaced towards the active conformation than for the corresponding wild-type. Absorption spectra, EPR and reduction potentials for the mutants are similar to those of the corresponding wild-type, although small shifts are observed in the spectra of the Tyr83Leu proteins. Based on these results, it is suggested that Photosystem 1 from spinach is capable of using both pea and spinach plastocyanin as an efficient electron donor and that the former even can stimulate the Photosystem 1 reduction. The origin of the stimulation is discussed in terms of differences in surface-exposed residues. Since the effects of the mutations are small, it can be concluded that electron transfer to Photosystem 1 does not occur via Tyr-83.Abbreviations cyt- cytochrome - IPTG- isopropyl--d-thiogalactopyranoside - P,P700- reaction-center chlorophyll - Pc- plastocyanin - PS 1- Photosystem 1 - SDS-PAGE- sodium dodecyl sulfate polyacrylamide gel electrophoresis - WT- wild-type  相似文献   

17.
The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A1, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A1 (Mr 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A1 polypeptide. The site of self ADP-ribosylation in the A1 subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A2 subunit in the holotoxin is at position 91.  相似文献   

18.
Cytochrome (cyt) b559, an integral membrane protein, is an essential component of the photosystem II (PSII) complex in the thylakoid membranes of oxygenic photosynthetic organisms. Cyt b559 has two subunits, alpha and beta, each with one predicted membrane spanning alpha-helical domain. The heme cofactor of this cytochrome is coordinated between two histidine residues. Each of the two subunit polypeptides of cyt b559 has one His residue. To investigate the influence of these His residues on the structure of cyt b559 and the PSII complex, we used a site directed mutagenesis approach to replace each His residue with a Leu residue. Introduction of these missense mutations in the transformable unicellular cyanobacterium, Synechocystis 6803, resulted in complete loss of PSII activity. Northern blot analysis showed that these mutations did not affect the stability of the polycistronic mRNA that encompasses both the psbE and the psbF genes, encoding the alpha and the beta subunits, respectively. Moreover, both of the single His mutants showed the presence of the alpha subunit which was 1.5 kd smaller than the same polypeptide in wild type cells. A secondary effect of such a structural change was that D1 and D2, two proteins that form the catalytic core (reaction center) of PSII, were also destabilized. Our results demonstrate that proper axial coordination of the heme cofactor in cyt b559 is important for the structural integrity of the reaction center of PSII.  相似文献   

19.
Lactococcus lactis prolidase preferably hydrolyzes Xaa-Pro dipeptides where Xaa is a hydrophobic amino acid. Anionic Glu-Pro and Asp-Pro dipeptides cannot be hydrolyzed at any observable rates and the hydrolysis of cationic Arg-Pro and Lys-Pro dipeptides is at about one tenth of the rate of Leu-Pro. It was hypothesized that the hydrophobic residues in the S1 site were responsible for this substrate specificity, thus the residues in the S1 site were substituted with hydrophilic residues. The substitution of Leu193 and Val302 revealed that these residues influenced the substrate specificity. The introduction of a cationic residue, L193R, allowed Asp-Pro to be utilized as a substrate at 37.0% of the rate of Leu-Pro, and the anionic mutation, V302D, yielded mutants that could hydrolyze Asp-Pro, Arg-Pro and Lys-Pro at 25.9 to 57.4% rates. Interestingly, these mutants of S1 site residues eliminated the allosteric behaviour of L. lactis prolidase that makes this enzyme unique among known prolidases. Results of pH dependency, thermal dependency, and molecular modelling suggested that these observed changes were due to the alteration of the interactions among catalytic zinc cations, Arg293, His296, and the mutated residues.  相似文献   

20.
The 5-, 12-, and 15-lipoxygenases contain a highly conserved sequence of the form His-(X)4-His-(X)4-His-(X)17-His-(X)8-His which represents a potential binding site for non heme iron to the protein. The importance of selected amino acids within this His cluster for the activity of human 5-lipoxygenase was investigated by site-directed mutagenesis using bacteria and insect cells expression systems. After single mutation of each of the 5 His residues at positions 363, 368, 373, 391, and 400 by Ser, Cys, or Lys, measurable levels of 5-lipoxygenase activity could be recovered in Escherichia coli only for the Ser363 and Cys363 mutants, with most amino acid substitutions causing a decrease in the levels of expression of the soluble protein. In contrast, 25-80% of soluble 5-lipoxygenase activity was recovered after the replacement of several of the hydrophobic amino acids in this region: Tyr384 by Ser or Phe; Phe394 by Trp and Val375 by Ala. Met436 could be replaced by Leu with little effect on 5-lipoxygenase activity or turnover inactivation half-time. High levels of mutant 5-lipoxygenases containing a Ser residue instead of His at each of the five positions were also expressed in Spodoptera frugiperda (Sf9) cells infected with recombinant baculovirus. The specific activity (58-75% of control) and the reaction time course of the Ser363, Ser391, and Ser400 mutants were comparable with that of native 5-lipoxygenase whereas inactive proteins were obtained for the Ser368 and Ser373 mutants. These results show that His368 and His373 residues are important for 5-lipoxygenase activity and that the other conserved His363, His391, His400, and Met436 residues are not crucial for the catalytic cycle or for the mechanism of self-inactivation of 5-lipoxygenase.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号